Gene methylation is an important regulator of gene expression. In particular, methylation at cytosine residues found in CpG di-nucleotide pairs in the promoter region of specific genes can contribute to many disease conditions through down regulation of gene expression. For example, aberrant methylation of tumour suppressor genes can lead to down regulation of these genes and is thus associated with the presence and development of many cancers. Patterns of aberrant gene methylation are often specific to the tissue of origin. Accordingly, detection of the methylation status of specific genes is of prognostic and diagnostic utility and can be used to both determine the relative stage of a disease and also to predict response to certain types of therapy.
O6-methylguanine-DNA methyltransferase (MGMT) is a cellular DNA repair protein that rapidly reverses alkylation (e.g. methylation) at the O6 position of guanine, thereby neutralizing the cytotoxic effects of alkylating agents used in therapy such as temozolomide (TMZ) and carmustine (1-3). It has been shown that epigenetic silencing of the MGMT gene by promoter silencing shuts down gene transcription (4, 5), and reflects a common alteration in primary human tumors that leads to MGMT deficiency (6). Epigenetic silencing of the MGMT gene has been shown to correlate with improved survival in several studies with glioma patients treated with alkylating agent therapy (7) and has been substantiated in two clinical trials (8, 9). The recent randomized clinical trial suggests that the MGMT-methylation status has a predictive value for benefit from the addition of the alkylating agent TMZ (9, 10). This finding has important clinical implications for stratified therapy (11). While this trial has established the new standard of care for glioblastoma patients (10), the benefit of the addition of TMZ chemotherapy was heavily weighted to patients whose tumors had a methylated MGMT promoter, with 46% still alive at 2 years, compared to only 14% of the patients with unmethylated MGMT (9). Hence, this epigenetic alteration in tumors can now be exploited in a diagnostic test to predict benefit from alkylating agent therapy for individualized management of patients. Beside glioblastoma, there is a published report that the MGMT methylation status may also predict benefit from alkylating agent containing therapy in patients with low grade glioma, oligodendroglioma, and diffuse large B-cell lymphoma (12, 13, 25)
Methylation-Specific PCR (MSP) with visualization of the results on a gel (gel-based MSP assay) is widely used to determine epigenetic silencing of genes (14), and in particular for testing MGMT promoter methylation in glioma (13, 15), although quantitative tests using other technologies have been developed (16-18). The nested gel-based MSP assay for MGMT has been used to establish the predictive value of the methylation status of the MGMT gene promoter in the clinical trials detailed above (8, 9). This methodology is highly sensitive and accurate, but has drawbacks for routine clinical use.
A number of fluorescence based technologies are available for real-time monitoring of nucleic acid amplification reactions. One such technology is described in U.S. Pat. No. 6,090,552 and EP 0912597 and is commercially known as Amplifluor®. This method is also suitable for end-point monitoring of nucleic acid amplification reactions.